The focus of this project is to decipher the mechanisms that govern the assembly of olfactory receptors from several subunits into homo- or heteromeric proteins and identify interactions with accessory proteins. Gaining insight into these assembly mechanisms and the analysis of the composition of olfactory receptors is crucial for a deeper understanding of how the reception of smell works. Smell is an important sense that helps us discriminate a myriad of nutrients and toxic substances. Mammalian and insect olfactory receptors are both seven-transmembrane-domain G-protein coupled receptors, but the membrane topology and the transduction mechanism have been shown to diverge. To understand the similarities and differences between olfactory receptors from different species will help to find common principles underlying olfaction. Olfactory receptors are thought to require dimerization to function. However, functional expression in heterologous cells depends on the co-expression of accessory proteins which help transport of the receptors to the cell surface. Some of these proteins are proposed to associate with the receptors to form multi-protein complexes. The experiments proposed intend to determine the number of subunits of the different receptor components, and the associations between them. At the beginning of the K99 phase, two vertebrate receptors and one insect olfactory receptor complex will be analyzed in Xenopus laevis oocytes. A novel method based on the visualization of fluorescently labeled single molecules allows the direct observation of associations between different proteins and counting of the subunit number of each protein in the complex. In the later stages of the K99 phase and in the R00 phase, we propose to use HEK293 cells as an expression system for the reconstitution of interactions between odorant receptors and accessory proteins. This will allow the analysis of receptors which cannot be expressed in Xenopus laevis oocytes without accessory proteins. RELEVANCE: Smell and taste are fundamental senses for the survival of any organism. These senses are mediated by odorant and taste receptors in specialized nerve cells. By deciphering how odorant receptors assemble from different subunits, we gain insight on how they interact with other components in the cell. Eventually, this will increase our understanding of how we sense smell and how this information is processed.